The overall goal of this application is to improve the diagnosis and treatment of bronchiolitis obliterans (BO) that occurs in the workplace as a result of exposure to artificial flavors. Workers in the food manufacturing industry are at significant rik for occupational airway disease due to exposure to commonly used artificial flavorings. In particular, diacetyl (DA) and pentanedione (PD), components used in butter flavoring, have been linked to the development of bronchiolitis obliterans (BO), an irreversible airway fibrosis. As a result, NIOSH NORA objectives include strategic goals focused on work-related airway diseases, specifically targeting studies of diacetyl and other potentially harmful artificial flavorings to improve workplace risk assessment (Goal 5.2.2) and define mechanisms of toxicity (Goal 5.1.3). This proposal directly addresses the NIOSH objectives for the Manufacturing Sector and Respiratory Disease Cross-sector. It is also consistent with the research-to-practice (r2p) goals by targeting an area prioritized as a major occupational health issue facing manufacturing workers. A primary goal of this project is to identify novel biomarkers of early lung injury after exposure to the artificial flavors DA and PD. Our overall hypothesis is that the protein signature from normal human bronchial epithelial (NHBE) cells exposed to DA or PD will provide a useful discovery platform for human biomarker development. By subsequently validating the most overexpressed proteins in a pre-clinical rodent BO model, we identify those targets that can best be translated into relevant blood or sputum tests for risk assessment in chemical flavoring exposed workers. As a secondary goal, we will use bioinformatic pathway analysis of the protein expression pattern of the NHBE cells in response to DA or PD to better define mechanisms of flavoring cellular toxicity, and determine which cellular pathways are activated in response to these agents. In preliminary studies in support of this application we have established that treatment of NHBE with DA or PD in concentrations up to 40 mm results in production of the cytokine interleukin (IL)-8 in a dose dependent manner, with minimal effects on cell viability. We demonstrated parallel increases in IL-8 in the lung flui in DA or PD-induced in vivo models of occupational BO in rodents. In the current application we will extend this preliminary data and 1) quantify the full spectrum of secreted proteins from cultured NHBE in response to DA or PD using state-of-the-art proteomic analysis technology, and apply bioinformatics analysis to determine pathways dysregulated by flavoring chemicals and 2) measure identified candidate proteins in the blood and lung fluid DA or PD-induced rodent models of occupational BO to prioritize selection of biomarkers for workplace studies. Our proposed unbiased discovery based approach is likely to deliver novel biomarkers that could improve early recognition of risk in the workplace and to provide new insights into the mechanisms of chemical flavoring toxicity that could suggest new actionable targets for BO prevention or treatment.